Okadaic acid (OA), tautomycin, calyculin A, and microcystin-LR are structurally diverse, tumor promoting natural products that apparently compete for binding to the same allosteric site on the catalytic subunits of protein phosphatases 1 and 2A (PP1 and PP2A, respectively). The low to sub-nanomolar inhibition of these protein phosphatases by the OA class of tumor promoters is associated with an array of physiological responses, most notably cellular transformation. This has stimulated the current widespread use of the OA-type inhibitors in studies of signal transduction, and cellular growth, regulation, and transformation. OA treatment has been reported to stimulate the expression of at least 10 widely different genes including the proto-oncogenes c-fos and c-jun, ornithine decarboxylase, glutathione-S-transferase, human collagenase, transin, urokinase, tyrosine amino transferase, phosphoenolpyruvate carboxylase, and the HIV-long terminal repeat. The sensitivities of PP1 and PP2A-like enzymes to okadaic acid are virtually identical in organisms as evolutionarily diverse as yeast, higher plants, and mammals. In spite of this, neither the mechanism of phosphatase inhibition, nor the structural basis for these different inhibitors to share the same allosteric binding site is known. Determination of the essential structural features that these tight binding protein phosphatase inhibitors have in common would aid the design of both inhibitor agonists and antagonists, and could help illuminate endogenous mechanisms of PP1 and PP2 regulation and control. The overall aim of this proposal is to determine the structural basis of PP1 and PP2A inhibition by the OA class of tumor promoters. This will involve determining the conformation of OA when bound to its receptor, and identifying the essential structural features of OA that are required for potent phosphatase inhibition. Previous studies attempting to address these issues have been limited by the availability of only a few naturally occurring and semi-synthetic derivatives of OA. Our approach to identify the OA pharmacophore involves the development of a new, efficient, and flexible total synthesis that can be readily adapted to the construction of a wide variety of analogs. OA is a C38 straight-chain fatty acid derivative containing 3 spiro-ketal ring systems each separated by linkages of 3 carbons. The first objective of this proposal is to synthesize conformationally constrained analogs of OA and test the hypothesis that the low energy solution phase conformation is involved in phosphatase binding and inhibition. Next, we will systematically examine which functional groups and structural features are necessary for inhibition. Additionally, we will synthesize photo affinity reagents based on the structure of OA and use them to covalently label, isolate, and identify the OA receptor to amino acid resolution. The synthetic probes will be assayed for their inhibition of PP2A catalytic subunit rho-nitrophenol phosphate phosphatase activity.